Novel Monoclonal Antibodies to Programmed DEATH (PD-1)

ABSTRACT

The present invention provides PD-1 monoclonal antibodies, particularly human monoclonal antibodies of PD-1, which specifically bind to PD-1 with high affinity and comprise a heavy chain and a light chain. The present invention further provides nucleic acid sequence encoding the antibodies of the invention, cloning or expression vectors, host cells and methods for expressing or isolating the antibodies. Immunoconjugates, therapeutic compositions comprising the antibodies of the invention are also provided. The invention also provides methods for treating various cancers with anti-PD-1 antibodies.

TECHNICAL FIELD

The present invention relates generally to antibodies of PD-1 andcompositions thereof, and immunotherapy in the treatment of humandisease using anti-PD-1 antibodies.

Background of the Invention

Increasing evidences from preclinical and clinical results have shownthat targeting immune checkpoints is becoming the most promisingapproach to treat patients with cancers. The protein Programmed Death 1(PD-1), an inhibitory member of the immunoglobulin super-family withhomology to CD28, is expressed on activated B cells, T cells, andmyeloid cells (Agata et al, supra; Okazaki et al (2002) Curr. Opin.Immunol. 14: 391779-82: Bennett et al. (2003) J Immunol 170:711-8) andhas a critical role in regulating stimulatory and inhibitory signals inthe immune system (Okazaki, Taku et al. 2007 International Immunology19:813-824). PD-1 was discovered through screening for differentialexpression in apoptotic cells (Ishida et al (1992) EMBO J 11:3887-95).

The PD-1 is a type I transmembrane protein that is part of the Ig genesuperfamily (Agata et al. (1996) bit Immunol 8:765-72) and the structureof PD-1 consists of one immunoglobulin variable-like extracellulardomain and a cytoplasmic domain containing an immunoreceptortyrosine-based inhibitory motif (ITIM) and an immunoreceptortyrosine-based switch motif (ITSM). Although structurally similar toCTLA-4, PD-1 lacks the MYPPPY motif that is critical for B7-1 and B7-2binding. PD-1 has two known ligands, PD-L1 (B7-H1, CD274) and PD-L2(B7-DC, CD273), which are cell surface expressed members of the B7family (Freeman et al (2000) J Exp Med 192: 1027-34; Latchman et al(2001) Nat Immunol 2:261-8; Carter et al (2002) Eur J Immunol32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, butdo not bind to other CD28 family members.

PD-1, as one of the immune-checkpoint proteins, is an inhibitory memberof the CD28 family expressed on activated B cells, T cells, and myeloidcells (Agata et al. supra; Okazaki et al. (2002) Curr Opin Immunol 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8) plays a major rolein limiting the activity of T cells that provide a major immuneresistance mechanism by which tumor cells escaped immune surveillance.PD-1 induces a state of anergy or unresponsiveness in T cells, resultingin the cells being unable to produce optimal levels of effectorcytokines. PD-1 may also induce apoptosis in T cells via its ability toinhibit survival signals. PD-1 deficient animals develop variousautoimmune phenotypes, including autoimmune cardiomyopathy and alupus-like syndrome with arthritis and nephritis (Nishimura et al.(1999) Immunity 11:141-51; Nishimura et al. (2001) Science 291:319-22).Additionally, PD-1 has been found to play a role in autoimmuneencephalomyelitis, systemic lupus erythematosus, graft-versus-hostdisease (GVHD), type I diabetes, and rheumatoid arthritis (Salama et al.(2003) J Exp Med 198:71-78: Prokunina and Alarcon-Riquelme (2004) HumMol Genet 13:R143; Nielsen et al. (2004) Lupus 11:510). In a murine Bcell tumor line, the ITSM of PD-1 was shown to be essential to blockBCR-mediated Ca²⁺-flux and tyrosine phosphorylation of downstreameffector molecules (Okazaki et al. (2001) PNAS 98: 13866-71).

The interaction of PD-1 expressed on activated T cells, and PD-L1expressed on tumor cells negatively regulates immune response and dampsanti-tumor immunity. PD-L1 is abundant in a variety of human cancers(Dong et al (2002) Nat. Med 8:787-9). Expression of PD-L1 on tumors iscorrelated with reduced survival in esophageal, pancreatic and othertypes of cancers, highlighting this pathway as a new promising targetfor tumor immunotherapy. Several groups have shown that the PD-1-PD-Linteraction exacerbates disease, resulting in a decrease in tumorinfiltrating lymphocytes, a decrease in T-cell receptor mediatedproliferation, and immune evasion by the cancerous cells (Dong et al.(2003) J. Mol. Med 81:281-7, Blank et al. (2005) Cancer Immunol.Immunother 54:307-314; Konishi et al. (2004) Clin. Cancer Res.10:5094-100). Immune suppression can be reversed by is inhibiting thelocal interaction of PD-1 with PD-L1, and the effect is additive whenthe interaction of PD-1 with PD-L2 is blocked as well.

Multiple agents targeting PD-1 pathway have been developed by severalpharmaceutical companies, such as Bristol-Myers Squibb (BMS), Merck,Roche and GlaxoSmithKline (GSK). Data from clinical trials demonstratedearly evidence of durable clinical activity and an encouraging safetyprofile in patients with various tumor types. Nivolumab, an anti-PD-1drug developed by BMS, is being put at center stage of thenext-generation field. Now in 6 late-stage studies, the treatmentspurred tumor shrinkage in three out of five cancer groups studied,including 18/6 of lung cancer patients (n=72), close to one third ofmelanoma patients (n=98) and 27% of patients with kidney cancer (n=33).Developed by Merck, Pembrolizumab is a humanized monoclonal IgG4antibody that acts against PD-1, which grabbed the FDA's newbreakthrough designation after impressive IB data came through for skincancer. The results from a phase IB study have shown an objectiveanti-tumor response in 51% of the cancer patients (n=85), and a completeresponse in 9% of the patients. Roche's experimental MPDL3280A(Atezolizumab) demonstrated an ability to shrink tumors in 29 of 140(21%) advanced cancer patients with various tumor sizes.

There are some spaces for improvement for antibody against PD-1 as atherapeutic agent. Most of monoclonal antibodies against PD-1 currentlytested in clinical trials are only against to human PD-1 which limitspreclinical in vivo assay and diminished efficacy owing to theimmunogenicity of the mouse-derived protein sequences. Humanizedantibody with cross-reactivity to mouse PD-1 overcome these shortagesand showed more tolerability and higher efficiency in vivo. Thus thereis still a need for novel anti-PD-1 antibody.

DISCLOSURE OF THE INVENTION

The present invention provides isolated antibodies, in particularmonoclonal antibodies or human monoclonal antibodies.

In one aspect, the present invention provides an antibody or antigenbinding fragment thereof that binds to an epitope of PD-1 comprisingamino acids at positions 128, 129, 130, 131 and 132 and at least one ofamino acids at positions 35, 64, 82, 83 of SEQ ID NO: 24.

The present invention also provides an antibody or antigen bindingfragment thereof that binds to an epitope of human and murine PD-1,wherein the epitope comprises amino acids at positions 128, 129, 130,131 and 132 of SEQ ID NO: 24.

The aforesaid antibody or the antigen binding fragment thereof, whereinthe murine PD-1 is mouse or rat PD-1.

The aforesaid antibody or antigen binding fragment thereof, wherein theantibody

a) binds to human PD-1 with a K_(D) of 2.15E-10 M or less; and

b) binds to mouse PD-1 with a K_(D) of 1.67E-08 M or less.

The aforesaid antibody, wherein the antibody

a) binds to human PD-1 with a K_(D) of 2.15E-10 M or less; and

b) binds to mouse PD-1 with a K_(D) of 1.67E-08 M or less, and

wherein the antibody exhibits at least one of the following properties:

a) binds to human PD-1 with a Ku of between 4.32E-10 M and 2.15E-10 Mand to mouse PD-1 with a K_(D) of between 5.39E-8 M and 1.67E-8 M;

b) does not substantially bind to human CD28, CTLA-4;

c) increases T-cell proliferation;

d) increases interferon-gamma production; or

e) increases interleukin-2 secretion.

The present invention provides an antibody or an antigen bindingfragment thereof, comprising an amino acid sequence that is at least 70%80%, 90% or 95% homologous to a sequence selected from a groupconsisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9, wherein theantibody specifically binds to PD-1.

The present invention provides an antibody or an antigen bindingfragment thereof, comprising an amino acid sequence selected from agroup consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 and 9, whereinthe antibody specifically binds to PD-1.

The present invention provides an antibody, or an antigen-bindingfragment thereof, comprising:

a) a variable region of a heavy chain having an amino acid sequence thatis at least 70%, 80%, 90% or 95% homologous to a sequence selected froma group consisting of SEQ ID NOs: 1 and 2; and

b) a variable region of a light chain having an amino acid sequence thatis at least 70%, 80%, 90/6 or 95% homologous to a sequence selected froma group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9,

wherein the antibody specifically binds to PD-1.

The present invention provides an antibody or an antigen bindingfragment thereof, comprising:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 1 and 2; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and9,

wherein the antibody specifically binds to PD-1.

In various embodiments, the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 3,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 3, wherein the antibodyspecifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 4,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 5,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 6,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 5,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 6,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 7,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 1; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 8,

wherein the antibody specifically binds to PD-1;

or the antibody comprises:

a) a variable region of a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 2; and

b) a variable region of a light chain having an amino acid sequenceselected from the group consisting of SEQ ID NO: 9,

wherein the antibody specifically binds to PD-1.

The sequence of the said antibody is shown in Table 1 and SequenceListing.

TABLE 1 Sequence of the antibody Clone ID SEQ ID NO Amino acid sequence1H6 Heavy chain 1 QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAILGYFDYWGQG TMVTVSS Light chain 3DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLE IK 2E5 Heavy chain 2QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGT MVTVSS Light chain 3DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFQGTKLE IK 2G4 Heavy chain 2QVQLVQSGAEVKKPGSSVKVSCKASGHIFITYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGT MVTVSS Light chain 4DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGSTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEI K 2C2 Heavy chain 2QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGT MVTVSS Light chainDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGATYL 5YWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEI K A6W Heavy chain 1QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAILGYFDYWGQG TMVTVSS Light chain 6DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGNTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLE IK 1G10 Heavy chain 1QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAILGYFDYWGQG TMVTVSS Light chain 5DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGATYLQVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEI K 2B1 Heavy chain 2VRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGT MVTVSS Light chain 6DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGNTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLE IK L1I Heavy chain 2QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGT MVTVSS Light chain 7DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGNTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHAPYTFGQGTKLE IK 5C4 Heavy chain 1QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAILGYFDYWGQG TMVTVSS Light chain 8DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGQTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHEPYTFGQGTKLEI K 8C10 Heavy chain 2QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGT MVTVSS Light chain 9DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGQTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHENYTFGQGTKLE IK

In another aspect, the invention provides an antibody or an antigenbinding fragment thereof, comprising a complementarity-determiningregion (CDR) having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-23,

wherein the antibody specifically binds to PD-1.

In another aspect, the invention provides an antibody, orantigen-binding fragment thereof., comprising: a heavy chain variableregion comprising CDR1, CDR2, and CDR3 sequences; and a light chainvariable region comprising CDR1, CDR2, and CDR3 sequences,

wherein the heavy chain variable region CDR3 sequence comprises asequence selected from a group consisting of SEQ ID NOs: 12 and 13, andconservative modifications thereof,

wherein the antibody specifically binds to PD-1.

Preferably, wherein the light chain variable region CDR3 sequence of theaforesaid antibody comprises an amino acid sequence selected from agroup consisting of SEQ ID NOs: 20, 21, 22 and 23, and conservativemodifications thereof.

Preferably, wherein the heavy chain variable region CDR2 sequence of theaforesaid antibody comprises an amino acid sequence selected from agroup consisting of amino acid sequences of SEQ ID NO: 11, andconservative modifications thereof.

Preferably, wherein the light chain variable region CDR2 sequence of theaforesaid antibody comprises an amino acid sequence selected from agroup consisting of amino acid sequences of SEQ ID NO: 19, andconservative modifications thereof.

Preferably, wherein the heavy chain variable region CDR1 sequence of theaforesaid antibody comprises an amino acid sequence selected from agroup consisting of amino acid sequences of SEQ ID NO: 10, andconservative modifications thereof.

Preferably, the antibody of this invention, wherein the light chainvariable region CDR1 sequence of the aforesaid antibody comprises anamino acid sequence selected from a group consisting of amino acidsequences of SEQ ID NO: 14, 15, 16, 17 and 18, and conservativemodifications thereof.

In more preferred embodiment, the invention provides an antibody, or anantigen-binding fragment thereof, wherein the antibody specificallybinds to PD-1 and comprises: a heavy chain variable region thatcomprises CDR1, CDR2, and CDR3 sequences; and a light chain variableregion that comprises CDR1, CDR2, and CDR3 sequences, wherein:

a) the heavy chain variable region CDR1 sequence comprises SEQ ID NO:10, and CDR2 sequence comprises an amino acid sequence selected from theSEQ ID NO: 11, CDR3 sequence comprises an amino acid sequence selectedfrom the group consisting of amino acid sequences of SEQ ID NOs: 12-13;

b) and the light chain variable region CDR1 sequence comprises an aminoacid sequence selected from the group consisting of amino acid sequencesof SEQ ID NOs: 14-18, CDR2 sequence comprises an amino acid sequenceselected from the group consisting of amino acid sequences of SEQ ID NO:19, CDR3 sequence comprises an amino acid sequence selected from thegroup consisting of amino acid sequences of SEQ ID NOs: 20-23.

A preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

d) a light chain variable region CDR1 comprising SEQ ID NOs: 14;

e) a light chain variable region CDR2 comprising SEQ ID NOs: 19;

f) a light chain variable region CDR3 comprising SEQ ID NOs: 20;

-   -   wherein the antibody specifically binds to PD-1.        Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NOs: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NOs: 13;

d) a light chain variable region CDR1 comprising SEQ ID NOs: 14;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 21;

-   -   wherein the antibody specifically binds to PD-1.        Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 13;

d) a light chain variable region CDR1 comprising SEQ ID NO: 15;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 21;

-   -   wherein the antibody specifically binds to PD-1.        Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 13;

d) a light chain variable region CDR1 comprising SEQ ID NO: 16;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 21;

wherein the antibody specifically binds to PD-1.Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

d) a light chain variable region CDR1 comprising SEQ ID NO: 17;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 21;

wherein the antibody specifically binds to PD-1.Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

d) a light chain variable region CDR1 comprising SEQ ID NO: 16;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 21;

wherein the antibody specifically binds to PD-1.Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 13;

d) a light chain variable region CDR1 comprising SEQ ID NO: 17;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 21;

-   -   wherein the antibody specifically binds to PD-1.        Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 13;

d) a light chain variable region CDR1 comprising SEQ ID NO: 17;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 22;

-   -   wherein the antibody specifically binds to PD-1.

Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

d) a light chain variable region CDR1 comprising SEQ ID NO: 18;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 23;

wherein the antibody specifically binds to PD-1.Another preferred antibody comprises:

a) a heavy chain variable region CDR1 comprising SEQ ID NO: 10;

b) a heavy chain variable region CDR2 comprising SEQ ID NO: 11;

c) a heavy chain variable region CDR3 comprising SEQ ID NO: 12;

d) a light chain variable region CDR1 comprising SEQ ID NO: 18;

e) a light chain variable region CDR2 comprising SEQ ID NO: 19;

f) a light chain variable region CDR3 comprising SEQ ID NO: 20;

wherein the antibody specifically binds to PD-1.

The CDR sequence of the said antibody is shown in Table 2 and SequenceListing.

TABLE 2 Sequence of the antibody SEQ ID SEQ ID SEQ ID Clone ID. NO CDR1NO CDR2 NO CDR3 1H6 Heavy 10 TYYIS 11 YINMGSGGTNYNEKFKG 12 LGYFDY chainLight 14 RSSQSLLDSDGGTYLY 19 LVSTLGS 20 MQLTHENYT chain 2E5 Heavy 10TYYIS 11 YINMGSGGTNYNEKFKG 13 IGYFDY chain Ligh 14 RSSQSLLDSDGGTYLY 19LVSTLGS 21 MQLTHWPYT chain 2G4 Heavy 10 TYYIS 11 YINMGSGGTNYNEKFKG 13IGYFDY chain Light 15 RSSQSLLDSDGSTYLY 19 LVSTLGS 21 MQLTHWPYT chain 2C2Heavy 10 TYYIS 11 YINMGSGGTNYNEKFKG 13 IGYFDY chain Light 16RSSQSLLDSDGATYLY 19 LVSTLGS 21 MQLTHWPYT chain A6W Heavy 10 TYYIS 11YINMGSGGTNYNEKFKG 12 LGYFDY chain Light 17 RSSQSLLDSDGNTYLY 19 LVSTLGS21 MQLTHWPYT chain 1G10 Heavy 10 TYYIS 11 YINMGSGGTNYNEKFKG 12 LGYFDYchain Light 16 RSSQSLLDSDGATYLY 19 LVSTLGS 21 MQLTHWPYT chain 2B1 Heavy10 TYYIS 11 YINMGSGGTNYNEKFKG 13 IGYFDY chain Light 17 RSSQSLLDSDGNTYLY19 LVSTLGS 21 MQLTHWPYT chain L11 Heavy 10 TYYIS 11 YINMGSGGTNYNEKFKG 13IGYFDY chain Ligh 17 RSSQSLLDSDGNTYLY 19 LVSTLGS 77 MQLTHAPYT chain 5C4Heavy 10 TYYIS 11 YINMGSGGTNYNEKFKG 12 LGYFDY chain Ligh 18RSSQSLLDSDGQTYLY 19 LVSTLGS 23 MQLTHEPYT chain 8C10 Heavy 10 TYYIS 11YINMGSGGTNYNEKFKG 12 LGYFDY chain Light 18 RSSQSLLDSDGQTYLY 19 LVSTLGS20 MQLTHENYT chain

The antibodies of the invention can be chimeric or humanized or humanantibody.

The antibodies of the invention can exhibit at least one of thefollowing properties:

a) binds to human PD-1 with a K_(D) of 2.15E-10 M or less and to mousePD-1 with a K_(D) of 1.67E-08 M or less;

b) does not substantially bind to human CD28, CTLA-4;

c) increases T-cell proliferation;

d) increases interferon-gamma production; or

e) increases interleukin-2 secretion.

In a further aspect, the invention provides a nucleic acid moleculeencoding the antibody, or antigen binding fragment thereof.

The invention provides a cloning or expression vector comprising thenucleic acid molecule encoding the antibody, or antigen binding fragmentthereof.

The invention also provides a host cell comprising one or more cloningor expression vectors.

In yet another aspect, the invention provides a process, comprisingculturing the host cell of the invention and isolating the antibody,wherein the antibody is prepared through immunization in SD rat withhuman PD-1 extracellular domain and mouse PD-1 extracellular domain.

The invention provides a transgenic mouse comprising humanimmunoglobulin heavy and light chain transgenes, wherein the mouseexpresses the antibody of this invention.

The invention provides hybridoma prepared from the mouse of thisinvention, wherein the hybridoma produces said antibody.

In a further aspect, the invention provides pharmaceutical compositioncomprising the antibody, or the antigen binding fragment of saidantibody in the invention, and one or more of a pharmaceuticallyacceptable excipient, diluent or carrier.

The invention provides an immunoconjugate comprising the said antibody,or antigen-binding fragment thereof in this invention, linked to atherapeutic agent.

Wherein, the invention provides a pharmaceutical composition comprisingthe said immunoconjugate and a pharmaceutically acceptable excipient,diluent or carrier.

The invention also provides a method for preparing an anti-PD-1 antibodyor an antigen-binding fragment thereof comprising:

-   -   (a) providing: (i) a heavy chain variable region antibody        sequence comprising a CDR1 sequence that is selected from a        group consisting of SEQ ID NO: 10, a CDR2 sequence that is        selected from a group consisting of SEQ ID NO: 11; and a CDR3        sequence that is selected from the group consisting of SEQ ID        NOs: 12 and 13; and/or (ii) a light chain variable region        antibody sequence comprising a CDR1 sequence that is selected        from the group consisting of SEQ ID NOs: 14, 15, 16, 17 and 18,        a CDR2 sequence that is selected from the group consisting of        SEQ ID NO: 19, and a CDR3 sequence that is selected from the        group consisting of SEQ ID NOs: 20, 21, 22 and 23; and    -   (b) expressing the altered antibody sequence as a protein.

The invention also provides a method of modulating an immune response ina subject comprising administering to the subject the antibody, orantigen binding fragment of any one of said antibodies in thisinvention.

The invention also provides the use of said antibody in the manufactureof a medicament for the treatment or prophylaxis of an immune disorderor cancer.

The invention also provides a method of inhibiting growth of tumor cellsin a subject, comprising administering to the subject a therapeuticallyeffective amount of the said antibody, or the said antigen-bindingfragment to inhibit growth of the tumor cells.

Wherein, the invention provides the method, wherein the tumor cells areof a cancer selected from a group consisting of melanoma, renal cancer,prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular malignant melanoma, uterine cancer, ovarian cancer, andrectal cancer.

Wherein, the invention provides the method, wherein the antibody is achimeric antibody or humanized antibody.

THE FEATURES AND ADVANTAGES OF THIS INVENTION

The inventors have generated a humanized antibody against PD-1 utilizingthe proprietary hybridoma technology. The antibodies reported in thisinvention have high binding affinity, specifically binding to both humanand mouse PD-1 protein without cross-family reactions; and potentmodulating immune responses, including enhancing T cell proliferationand increasing cytokine IFN-γ and interleukin-2 production.

New anti-PD-1 antibodies binding to mouse PD-1 are derived from immunedrats, which overcomes the disadvantage that is anti-PD-1 antibodies cannot be used in pre-clinical mouse model; and the humanized level isclose to 100% after sequence humanization, greatly reducing the adverseeffects of drugs used in the human body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show graphs of hybridoma antibodies binding to cellsurface human and mouse PD-1. FIG. 1A shows binding to human PD-1. FIG.1B shows binding to mouse PD-1.

FIG. 2 shows the result from first around mutagenesis library screen.Sequence and analysis mutation on high affinity clones for the secondaround mutation.

FIGS. 3A, 3B and 3C show the results of cross-species test by FACS. FIG.3A shows binding to human PD-1 transfected CHO-S cells. FIG. 3B showsbinding to mouse PD-1 transfected 293F cells. FIG. 3C shows binding toactivated cynomolgus PBMC. Note: the isotype was human IgG4 kappa. Thesame below.

FIG. 4 shows the result of cross-species test by ELISA. FIG. 4A showsbinding to human PD-1. FIG. 4B shows binding to mouse PD-1. FIG. 4Cshows binding to cynomolgus PD-1.

FIG. 5 shows the result of cross-family test. The anti-PD-1 antibodiesbind specifically to human PD-1, but not to CD28 and CTLA-4.

FIG. 6A shows the result of anti-PD-1 antibodies blocking human PD-L1binding to PD-1 transfected CHO-S cells. FIG. 6B shows the result ofanti-PD-1 antibodies blocking mouse PD-L1 binding to PD-1 transfected293F cells.

FIG. 7 shows that the anti-PD-1 antibodies could block human PD-L2binding to PD-1.

FIG. 8A-8B show the results of epitope binning assay suggesting that theanti-PD-1 antibodies are in the same or close epitope bin as benchmarkantibodies. FIG. 8A shows binning against WBP305BMK1 (U.S. Pat. No.9,084,776). FIG. 8B shows binning against Keytruda (U.S. Pat. No.8,168,757).

FIG. 9 shows the cross-reactivity of anti-PD-11 antibodies withhuman/mouse PD-1. 2 μg/mL of each antibody were coated at 96-well plateovernight and incubated with hPD-1/mPD-1-His protein, then HRP-anti-Hisantibody were added for detection.

FIG. 10 shows the Hot spot residues mapped on hPD-1 structure. (A).hPD-L1 binding site. Data were obtained from the literature Zak et al.2015. (B-C). Binding site of antibody W3052_r16.88.9 and Keytruda,respectively. Data were from table 8. Colors on the pictures are to helpdistinguish the differences between epitopes.

FIG. 11 shows comparison between human and murine PD-1. Their obviousstructural differences (BC loop and C′D loop (or C″ strand on mPD-1))were marked in orange color. (A). Structures of hPD-1 (PDB code 4ZQK).The missing loop (Asp85-Asp92) were remolded based on its NMR structures(PDB code 2M2D). (B). Structure of mPD-1 (PDB code 3BIK).

FIG. 12A-12C show the results of human allo-MLR demonstrating theanti-PD-1 antibodies can enhance the function of human CD4⁺ T cell. FIG.12A shows anti-PD-1 antibodies increase IL-2 secretion in adose-dependent manner. FIG. 12B shows anti-PD-1 antibodies increaseIFN-γ secretion in a dose-dependent manner. FIG. 12C shows anti-PD-1antibodies increase CD4⁺ T cells proliferation in a dose-dependentmanner.

FIG. 13A-13C show the results of mouse allo-MLR demonstrating that theanti-PD-1 antibodies can enhance the function of mouse CD4⁺ T cell.

FIG. 13A shows anti-PD-1 antibodies increase IL-2 secretion in adose-dependent manner. FIG. 13B shows anti-PD-1 antibodies increaseIFN-γ secretion in a dose-dependent manner. FIG. 13C shows anti-PD-1antibodies increase CD4⁺ T cells proliferation in a dose-dependentmanner.

FIG. 14A-14B show the results of human allo-MLR demonstrating theanti-PD-1 antibodies can enhance the function of human CD4⁺ T cell. FIG.14A shows anti-PD-1 antibodies increase IFN-γ secretion in adose-dependent manner. FIG. 14B shows anti-PD-1 antibodies increase CD4⁺T cells proliferation in a dose-dependent manner.

FIGS. 15A and 15B demonstrate that the anti-PD-1 antibodies can reversethe suppressive function of Tregs. FIG. 15A shows anti-PD-1 antibodiescan restore the IFN-γ secretion. FIG. 15B shows anti-PD-1 antibodies canrestore the T-cell proliferation.

FIG. 16 shows the result of ADCC test demonstrating the anti-PD-1antibodies do not mediate ADCC activity on activated CD4⁺ T cells.

FIG. 17 shows the result of CDC test demonstrating the anti-PD-1antibodies do not mediate CDC activity on activated CD4+ T cells.

FIG. 18 shows body weight changes in syngeneic tumor nude mice modelafter treatment of 2E5. The data point represents the average bodyweight; error bars represent the standard error (SEM).

FIG. 19 shows relative weight changes (%). Relative change in bodyweight was calculated based on body weight at the start of theadministration. The data point represents the average body weight; errorbars represent the standard error (SEM).

FIG. 20 shows tumor growth curve in CloudmanS91 syngeneic tumor nudemice model after treatment of 2E5. The data point represents the averagebody weight; error bars represent the standard error (SEM).

FIG. 21 shows survival curve in CloudmanS91 syngeneic tumor nude micemodel after treatment of 2E5.

DETAILED DESCRIPTION

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “Programmed Death 1”, “Programmed Cell Death 1”, “ProteinPD-1”, “PD-1”, “PD1”, “PDCD1”, “hPD-1” and “hPD-F” are usedinterchangeably, and include variants, isoforms, species homologs ofhuman PD-1, and analogs having at least one common epitope with PD-1.

The term “antibody” as referred to herein includes whole antibodies andany antigen-binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a protein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen-binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen.

The term “antibody,” as used in this disclosure, refers to animmunoglobulin or a fragment or a derivative thereof, and encompassesany polypeptide comprising an antigen-binding site, regardless whetherit is produced in vitro or in vivo. The term includes, but is notlimited to, polyclonal, monoclonal, monospecific, polyspecific,non-specific, humanized, single-chain, chimeric, synthetic, recombinant,hybrid, mutated, and grafted antibodies. The term “antibody” alsoincludes antibody fragments such as Fab, F(ab′)2, Fv, scFv, Fd, dAb, andother antibody fragments that retain antigen-binding function, i.e., theability to bind PD-1 specifically. Typically, such fragments wouldcomprise an antigen-binding fragment.

The terms “antigen-binding fragment,” “antigen-binding domain,” and“binding fragment” refer to a part of an antibody molecule thatcomprises amino acids responsible for the specific binding between theantibody and the antigen. In instances, where an antigen is large, theantigen-binding fragment may only bind to a part of the antigen. Aportion of the antigen molecule that is responsible for specificinteractions with the antigen-binding fragment is referred to as“epitope” or “antigenic determinant.”

An antigen-binding fragment typically comprises an antibody light chainvariable region (VL) and an antibody heavy chain variable region (VII),however, it does not necessarily have to comprise both. For example, aso-called Fd antibody fragment consists only of a VII domain, but stillretains some antigen-binding function of the intact antibody.

In line with the above the term “epitope” defines an antigenicdeterminant, which is specifically bound/identified by a bindingfragment as defined above. The binding fragment may specifically bindto/interact with conformational or continuous epitopes, which are uniquefor the target structure, e.g. the human and murine PD-1. Aconformational or discontinuous epitope is characterized for polypeptideantigens by the presence of two or more discrete amino acid residueswhich are separated in the primary sequence, but come together on thesurface of the molecule when the polypeptide folds into the nativeprotein/antigen. The two or more discrete amino acid residuescontributing to the epitope are present on separate sections of one ormore polypeptide chain(s). These residues come together on the surfaceof the molecule when the polypeptide chain(s) fold(s) into athree-dimensional structure to constitute the epitope. In contrast, acontinuous or linear epitope consists of two or more discrete amino acidresidues, which are present in a single linear segment of a polypeptidechain.

The term “binds to an epitope of PD-1” refers to the antibodies havespecific binding for a particular epitope of PD-1, which may be definedby a linear amino acid sequence, or by a tertiary, i.e.,three-dimensional, conformation on part of the PD-1 polypeptide. Bindingmeans that the antibodies affinity for the portion of PD-1 issubstantially greater than their affinity for other relatedpolypeptides. The term “substantially greater affinity” means that thereis a measurable increase in the affinity for the portion of PD-1 ascompared with the affinity for other related polypeptides. Preferably,the affinity is at least 1.5-fold, 2-fold, 5-fold 10-fold, 100-fold,10³-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold or greater for the particularportion of PD-1 than for other proteins. Preferably, the bindingaffinity is determined by enzyme-linked immunoabsorbent assay (ELISA),or by fluorescence-activated cell sorting (FACS) analysis or surfacePlasnon resonance (SPR). More preferably, the binding specificity isobtained by fluorescence-activated cell sorting (FACS) analysis.

The term “cross-reactivity” refers to binding of an antigen fragmentdescribed herein to the same target molecule in human and murine (mouseor rat). Thus, “cross-reactivity” is to be understood as an interspeciesreactivity to the same molecule X expressed in different species, butnot to a molecule other than X. Cross-species specificity of amonoclonal antibody recognizing e.g. human PD-1, to a murine (mouse orrat) PD-1, can be determined, for instance, by FACS analysis.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc. Except when noted,the terms “patient” or “subject” are used interchangeably.

The terms “treatment” and “therapeutic method” refer to both therapeutictreatment and prophylactic/preventative measures. Those in need oftreatment may include individuals already having a particular medicaldisorder as well as those who may ultimately acquire the disorder.

EXAMPLES Example 1: Research Materials Preparation 1. ImmunogenGeneration.

DNAs encoding the ECD or full length of PD-1 and PD-L1 were synthesizedand inserted into the expression vector pcDNA3.3. Max-prep the plasmidDNAs and the inserted DNA sequences were verified by sequencing. Fusionproteins PD-1 ECD and PD-L1 ECD containing various tags, including humanFc, mouse Fc and His tags, were obtained by transfection of human PD-1ECD gene into CHO-S or HEK293 cells. After 5 days, supernatants wereharvested from the culture of transient transfected cells. The fusionproteins were purified and quantified for usage of immunization andscreening.

2. Stable Cell Lines Establishment

In order to obtain tools for antibody screening and validation, wegenerated PD-1 and PD-L1 transfecting cell lines. Briefly, CHO—K1 or293F cells were transfected with pcDNA3.3 expression vector containingfull-length PD-1 or PD-L1 using Lipofectamine 2000 Transfection kitaccording to manufacturer's protocol. 48-72 hours post transfection; thetransfected cells were cultured in medium containing Blasticidin or G418to select the cells that had PD-1 or PD-L1 genes stably incorporatedinto their genomic DNAs. Meanwhile the cells were checked for interestedgenes PD-1 and PD-L1 expression. Once the expression verified, singleclones of interested were picked by limited dilution and scaled up tolarge volumes. The established monoclonal cell lines were thenmaintained in medium containing lower dose of antibiotics Blasticidin orG418.

Example 2: Antibody Hybridoma Generation 1. Immunization

Female SD rats, at 6-8 weeks of age, were immunized with 10 μg/animal ofhuman PD-1 ECD protein and 10 μg/animal of mouse PD-1 ECD protein inTiterMax by footpad injection for prime, and were boosted twice a weekwith human PD-1 ECD protein or mouse PD-1 ECD protein in Aluminiumalternately. The serum antibody titers were measured by ELISA or FACSevery two weeks.

2. Cell Fusion

When the serum antibody titer was sufficiently high, rats were given afinal boost with both human and mouse PD-1 ECD protein in the equalvolume of D-PBS (Dulbecco's Phosphate Buffered Saline) without adjuvant.The cell fusion was performed as follows: preparing myeloma cells SP2/0,myeloma cells were thawed the week before the fusion, and were split at1:2 each day until the day before the fusion to keep in logarithmicgrowth. B lymphocytes isolated from lymph node of immunized SD rats werecombined with myeloma cells (at 1:1 ratio). The cells were treated withTrypsin and the reaction was stopped by FBS. Cell mixture was thenwashed and re-suspended in ECF solution at 2×10⁶ cells/ml for ECF. Afterelectronic cell fusion (BTX2000), cell suspension from the fusionchamber was immediately transferred into a sterile tube containing moremedium, and incubated for at least 24 hours in a 37° C. incubator. Thecell suspension was then mixed and transferred into 96-well plates(1×10⁴ cells/well). The 96-well plates were cultured at 37° C., 5% CO₂,and were monitored periodically. When the clones were big enough (after7-14 days), 100 μL of supernatant were transferred from the tissueculture plates to 96-well assay plates for antibody screening.

3. First, Second and Confirmation Screen of Hybridoma Supernatants

ELISA assay was used as first screen method to test the binding ofhybridoma supernatants to human or mouse PD-1 protein. Briefly, plates(Nunc) were coated with human or mouse PD-1 ECD at 1 μg/ml overnight at4° C. After blocking and washing, the hybridoma supernatants were loadedto the coated plates and incubated at room temperature for 1 h. Theplates were then washed and subsequently incubated with secondaryantibody goat anti rat IgG Fc HRP (Bethyl) for 1 h. After washing, TMBsubstrate was added and the reaction was stopped by 2M HCl. Theabsorbance at 450 nm was read using a microplate reader (MolecularDevice).

In order to confirm the native binding of anti-PD-1 antibodies onconformational PD-1 molecules expressed on cell membrane, FACS analysiswas performed using PD-1 transfected cell lines as second screening.CHO-S cells expressing human PD-1 or 293F cells expressing mouse PD-1were transferred into 96-well U-bottom plates (Corning) at a density of1×10⁺ cells/well. The hybridoma supernatants were then added andincubated with the cells for 1 h at 4° C. After washing with 1x PBS/I %BSA, the secondary antibody goat anti rat FITC (Jackson ImmunoResearchLab) was applied and incubated with cells at 4° C. in the dark for 1 h.The cells were then washed and resuspended in 1>PBS/l % BSA or fixedwith 4% paraformldehyde, and analyzed by flow cytometery (BD) and FlowJosoftware. Antibody binding to parental CHO-S or 293F cell line was usedas negative control, respectively.

To select potential antagonistic hits, selected antibodies were testedfor their ability to block the binding of the ligand PD-L1 to PD-1transfected cells by FACS analysis. CHO-S cells expressing human PD-1 or293F cells expressing mouse PD-1 were transferred into 96-well U-bottomplates (BD) at a density of 1×10³ cells/well. Hybridoma supernatantswere added and incubated with the cells at 4° C. for 1 h. After washing,mouse Fc fusion-human PD-L1 protein or mouse Fc fusion-mouse PD-L1protein was added and incubated at 4° C. for 1 h. The secondary antibodygoat anti mouse IgG Fc FITC antibody (no cross-reactivity to rat IgG Fc,Jackson ImmunoResearch Lab) was incubated with cells at 4° C. in thedark for 1 h. The cells were then washed and resuspended in 1×PBS/1% BSAor fixed with 4% paraformldehyde, and analyzed by flow cytometery (BD)and FlowJo software.

FIG. 1 Shows Graphs of 16 Hybridoma Antibodies Binding to Cell Surfacehuman and mouse PD-1. FIG. 1A shows binding to human PD-1. FIG. 1B showsbinding to mouse PD-1.

4. Hybridoma Subcloning

Once specific binding and blocking activity were verified through firstand confirmation screening, the positive hybridoma cell lines were usedfor subcloning. Briefly, for each hybridoma cell line, cells werecounted and diluted to give 5 cells, 1 cell or 0.5 cell per 200 μLcloning medium. The cell suspension was plated 200 μL/well into 96-wellplates, one plate at 5 cells/well, one plate at 1 cell/well and fourplates at 0.5 cell/well. Plates were cultured at 37° C., 5% CO₂, tillthey were ready to be screened by binding ELISA or FACS as describedabove. The ESN of selected single clones were collected, and theantibodies were purified for further characterization.

5. Subtypes Testing

50 μL of goat anti-rat IgG1, IgG2a, IgG2b, IgG2c, IgG or IgM antibodies(1 μg/mL) were coated in microtiter plates (Nunc) per well overnight.After blocking. 50 μL of hybridoma supernatant samples were added toeach well, incubated for 2 hours at room temperature. Goat anti-rat IgGkappa or IRP labeled lambda light chain secondary antibody (Bethyl) is adetection antibody. Using TMB substrate for color, the reaction was thenquenched with 2 M HCl. The value of absorbs light at 450 nm is readusing a microplate reader (Molecular Device).

Table 3 shows the subtype results of 16 hybridoma antibodies. 7antibodies are polyclonal antibodies, and 9 antibodies are IgG2a kappasubtype. Considering the needs of anti-PD-1 antibody to avoid the roleof ADCC and CDC in vivo, the humanized antibody will be built as humanIgG4 kappa subtype.

TABLE 3 Subtypes of the hybridoma antibodies kappa Number antibody IgG1IgG2a IgG2b IgM 1 W3052_r16.6.25 − − + weak 2 W3052_r16.14.6 + + − − 3W3052_r16.14.16 − + − + 4 W3052_r16.16.14 + + − + 5 W3052_r16.53.26 + +− − 6 W3052_r16.68.22 − + − − 7 W3052_r16.68.41 − + − − 8 W3052_r16.81.3− − + + 9 W3052_r16.88.9 − + − − 10 W3052_r16.88.21 weak + − − 11W3052_r16.88.29 − weak − − 12 W3052_r16.88.32 − + − − 13W3052_r16.114.2 + 14 W3052_r16.114.8 − + − − 15 W3052_r16.114.15 − + − −16 W3052_r16.114.39 − + − −

Example 3: Antibody Hybridoma Cell Sequence and Humanized AntibodyMolecules Construction and Affinity Maturation 1. Antibody HybridomaCell Sequence

RNAs were isolated from monoclonal hybridoma cells with Trizol reagentThe VH and VL of PD-1 chimeric antibodies were amplified as follows: RNAis first reverse transcribed into cDNA using a reverse transcriptase asdescribed here.

Reaction System (20 μL)

10 × RT Buffer 2.0 μL 25 × dNTP Mix (100 mM) 0.8 μL 10 × RT RandomPrimers/oligodT/specific primer 2.0 μL MultiScribe ™ ReverseTranscriptase 1.0 μL RNase Inhibitor 1.0 μL RNA 2 μg Nuclease-free H₂Oto 20.0 μL

Reaction Condition

Step 1 Step 2 Step 3 Step 4 Temperature (° C.) 25 37 85 4 Time 10 min120 min 5 ∞

The resulting cDNA was used as templates for subsequent PCRamplification using primers specific for interested genes. The PCRreaction was done as follows:

cDNA 1 μL Ex PCR buffer 5 μL dNTP 2 μL ExTaq 0.5 μL P1 (25 pM) 0.5 μL P2(25 pM) 0.5 μL ddH₂O 40.5 μL

Reaction Condition:

94° C. 3 min 94° C. 30 s {close oversize brace} 30 cycles 60° C. 30 s72° C. 1 min 72° C. 10 min

The resulting PCR product (10 μL) was ligated with pMD18-T vector. Top10competent cells were transformed with 10 μL of the ligation product.Positive clones were checked by PCR using M13-48 and M13-47 primersfollowed by sequencing.

2. Humanized Antibody Molecule Construction

The rat anti-PD-1 antibody from hybridomas were selected and humanizedaccording to the high affinity and specificity of anti-PD-1 antibodybinding to PD-1, improving the homology with human antibody sequence.The said humanized usage is called as CDR-grafting technique. Thevariable region gene of antibody such as FR regions and CDR regions weredivided by KABAT system and IMGT system. In antibody database, based onthe alignments of binding sequence homology and structural similarity,the gene of murine region FR1-3 was replaced by humanized variableregion FR1-3, region FR4 of the murine gene was replaced by humanizedFR4 region derived from JH and JK genes which had the most similarstructures. After verifying the template sequence and codonoptimization, the heavy chain variable region and light chain variableregion were synthesized and cloned into the expression vector, and thenexpressing the humanized antibody.

According to the binding ability to cell surface human and mouse PD-1,W3052_r16.88.9 and W3052_r16.81.3 was selected for humanization. Table.2shows the analysis of humanization scores. The clonesW3052-16.88-z9-IgG4 (42720) was selected for affinity maturationconsidering all these factors such as better affinity and humanizationscores (Table.4).

TABLE 4 Humanization Lead antibody FR1 FR2 FR3 FR4 scoreW3052_r16.88.hAb140798 WBP305_r16.88- IGHV1-69*06 100% 100% 93.30%  IGHJ3*01 100% 99.16% hVH1-m WBP305_r16.88- IGKV2-29*02 100% 100% 100%IGKJ2*01 100% VL1 W3052_r16.88.hAb240764 WBP305_r16.88- IGHV1-69*06 100%(IGHV1- 93.30%   IGHJ3*01 100% 99.16% hVH2 8*01) 100% WBP305_r16.88-IGKV2-29*02 100% 100% 100% IGKJ2*01 100% VL1 W3052_r16.88.hAb340766WBP305_r16.88- IGHV1-69*06 100% 85.70%   93.30%   IGHJ3*01 100% 97.38%hVH3 WBP305_r16.88- IGKV2-29*02 100% 100% 100% IGKJ2*01 100% VL1W3052_r16.88.hAb440770 WBP305_r16.88- IGHV1-69*06 100% 100% 93.30%  IGHJ3*01 100% 99.16% hVH1 WBP305_r16.88- IGKV2-30*01 100% 100% 100%IGKJ2*01 100% VL2 W3052_r16.88.hAb540773 WBP305_r16.88- IGHV1-69*06 100%(IGHV1- 93.30%   IGHJ3*01 100% 99.16% hVH2-m 8*01) 100% WBP305_r16.88-IGKV2-30*01 100% 100% 100% IGKJ2*01 100% VL2 W3052_r16.88.hAb640800WBP305_r16.88- IGHV1-69*06 100% 85.70%   93.30%   IGHJ3*01 100% 97.38%hVH3-m WBP305_r16.88- IGKV2-30*01 100% 100% 100% IGKJ2*01 100% VL2W3052_r16.88- WBP305_r16.88- IGHV1-69*06 100% 100% 100% IGHJ3*01 100% 100% z7-IgG442691 hVH1 WBP305_r16.88- IGKV2-29*02 100% 100% 100%IGKJ2*01 100% VL1 W3052_r16.88- WBP305_r16.88- IGHV1-69*06 100% (IGHV1-100% IGHJ3*01 100%  100% z8-IgG442715 hVH2 8*01) 100% WBP305_r16.88-IGKV2-30*01 100% 100% 100% IGKJ2*01 100% VL2 W3052_r16.88-WBP305_r16.88- IGHV1-69*06 100% 85.70%   100% IGHJ3*01 100% 98.20%z9-IgG442720 hVH3 WBP305_r16.88- IGKV2-30*01 100% 100% 100% IGKJ2*01100% VL2 W3052_r16.81.hAb140779 WBP305_r16.81- IGHV3-7*03 100% 100% 100%IGHJ1*01 100%  100% VH1 WBP305_r16.81- IGKV1D- 100% 100% 100% IGKJ4*01100% VL1 16*02 W3052_r16.81.hAb240781 WBP305_r16.81- IGHV3-9*01 100%100% 100% IGHJ1*01 100%  100% VH2 WBP305_r16.81- IGKV1D- 100% 100% 100%IGKJ4*01 100% VL1 16*02 W3052_r16.81.hAb340784 WBP305_r16.81- IGHV3-7*03100% 100% 100% IGHJ1*01 100%  100% VH1 WBP305_r16.81- IGKV1-39*01 100%100% 100% IGKJ4*01 100% VL2 W3052_r16.81.hAb440787 WBP305_r16.81-IGHV3-9*01 100% 100% 100% IGHJ1*01 100%  100% VH2 WBP305_r16.81-IGKV1-39*01 100% 100% 100% IGKJ4*01 100% VL2

3. Affinity Maturation.

Each amino acid of three complementary-determining regions (VH CDR3, VKCDR1, and VK CDR3) of parental clone was individually mutated to other20 amino acids using a hybridization mnutagenesis method. DNA primerscontaining a NNS codon encoding twenty amino acids were used tointroduce mutation to each targeted CDR position. The individualdegenerate primers were used in hybridization mutagenesis reactions.Briefly, each degenerate primer was phosphorylated, and then used in a10:1 ratio with uridinylated ssDNA. The mixture was heated to 85° C. for5 minutes then cooled down to 55° C. over 1 hour. Thereafter, T4 ligaseand T4 DNA polymerase were added and mix was incubated for 1.5 hours at37° C. Synthesis products for VII and VL CDRs were pooled respectively.Typically, 200 ng of the pooled library DNA was electroporated into BL21for plaque formation on BL21 bacterial lawn or for production of scFvfragments.

The primary screen consisted of a single point ELISA (SPE) assay whichwas carried out using periplasmic extract (PE) of bacteria grown in96-well plates (deep well). Briefly, this capture ELISA involved coatingindividual wells of a 96-well Maxisorp Immunoplate with anti-c-mycantibody in coating buffer (200 mM Na₂CO₃/NaHCO₃) at pH 9.2 overnight at4° C. The next day, the plate was blocked with Casein for 1 h at roomtemperature. scFv PE was then added to the plate and incubated at roomtemperature for 1 hr. After washing, biotinylated antigen protein wasadded to the well and the mixture was incubated for 1h at roomtemperature. This was followed by incubation with Streptavidin-HRPconjugate for 1h at room temperature. HRP activity was detected with TMBsubstrate and the reaction was quenched with 2 M HCl. Plates were readat 450 nm. Clones exhibiting an optical density (OD) signal at 450 nmgreater than the parental clone were picked and re-assayed by ELISA (asdescribed above) in duplicate to confirm positive results. Clones thatrepeatedly exhibited a signal greater than that of the parental antibodywere sequenced. The scFv protein concentration of each clone that had aCDR change was then determined by a quantitative scFv ELISA, where ascFv with known concentration was used as a reference. The scFv proteinconcentration was determined by comparing the ELISA signals with signalsgenerated by the reference scFv. The binding assay was repeated oncemore for all positive variants under normalized scFv concentration inorder to determine the relative binding affinity of the mutant scFv andthe parental antibody.

The point mutations in VH and VL determined to be beneficial for bindingto antigen were further combined to gain additional binding synergy. Thecombinatorial mutants were expressed as scFv and screened using thecapture ELISA. Clones exhibiting an OD signal at 450 nm greater than theparental clone were sequenced and further confirmed by binding ELISA asdescribed above.

After affinity maturation, a total of 10 humanized antibodies (2E5, 2G4,1G10, 2C2, 2B1, 8C10, 1H6, 5C4, A6W and L1I) were obtained. FIG. 2showed the result from first round mutagenesis library screen. Sequenceand affinity data of 10 humanized antibodies in human, cynomolgusmonkeys and mice were shown in Table 5.

Table.5 showed the result from second round mutagenesis library screen.The clones 1H6, 2E5, 2G4 and 2C2 were selected for further analysis.

TABLE 5 Bmax Kd Bmax Kd Bmax Kd (Cyno- (Cyno- Name VHCDR3 VKCDR1 VKCDR3(Human) (Human) (Mouse) (Mouse) molgus) gus) 2E5 1 LDSDGGTYLYWMOLTHWPYTFGQ 3.279 0.0675 0.4696 0.0443 1.72 0.1088 2G4 1 LDSDGSTYLYWMQLTHWPYTFGQ 3.371 0.0708 0.4793 0 0426 1.718 0.1057 1G10 1 LDSDGATYLYWMQLTHWPYTFGQ 2.600 0.0711 0.2997 0.0718 1.082 0.1224 2C2 1 LDSDGATYLYWMQLTHWPYTFGQ 3.175 0.082 0.416 0.049 1.668 0.116 2B1 1 LDSDGNTYLYWMQLTHWPYTFGQ 3.019 0.0912 0.3393 0.0346 1.207 0.1142 8C10 1 LDSDGQTYLYWMQLTHENYTFGQ 2.307 0.104 0.437 0.038 1.109 0.280 1H6 1 LDSDGGTYLYWMQLTHWPYTFGQ 3.348 0.1114 0.2213 0.0466 0.3171 0.0977 5C4 1 LDSDGQTYLYWMQLTHEPYTFGQ 2.649 0.236 0.164 0 027 0.258 0.292 A6W 1 LDSDGNTYLYWMQLTHWPYTFGQ 2.571 0.2885 0 1736 0.0294 0.0787 0.0016 L11 1 LDSDGNTYLYWMQLTHAPYTFGQ 1.048 1.8370 0.1048 0.0050 0.05175 N/A

4. Antibody Purification.

The vector containing affinity matured humanized antibody weretransfected into 293F cells for antibody production and expression.Antibodies in the supernatant of 293F cells were purified using ProteinA affinity chromatography.

EXAMPLE 4: Characterization of Humanized Antibody 1. Cross-Reactivity toHuman, Cynomolgus and Mouse PD-1 (Cross-Species) 1.1 FACS.

Cross-reactivity was measured by FACS and ELISA. For FACS, the anti-PD-1antibodies were tested binding to cell surface human, mouse andcynomolgus PD-1 as described in Example 2.3.

FIG. 3 showed the results of cross-species test by FACS. FIG. 3A showedbinding to human PD-1 transfected CHO-S cells. The antibodies can bindspecifically to the human PD-1 with EC50 of 2.20-2.78 nM. FIG. 3B showedbinding to mouse PD-1 transfected 293F cells. The antibodies can bindspecifically to the mouse PD-1 with EC50 of 11.8-15.1 nM. FIG. 3C showedbinding to activated cynomolgus PBMC in a dose dependent way. Theisotype was human IgG4 kappa. The same below.

1.2 Cross-Reactivity to Human, Cynomolgus and Mouse PD-1 (Cross-Species)

For ELISA, plates (Nunc) were coated with human, cynomolgus or mousePD-1 (Sino Biological) at 1 μg/ml overnight at 4° C. After blocking andwashing, antibodies were serially diluted in blocking buffer and addedto the plates and incubated at room temperature for 1 h. The plates werethen washed and subsequently incubated with secondary antibody goat antihuman IgG HRP (Bethyl) for 1 h. After washing, TMB substrate was addedand the reaction was stopped by 2 M HCl. The absorbance at 450 nm wasread using a microplate reader (Molecular Device).

FIG. 4 showed the result of cross-species test by ELISA. FIG. 4A showedbinding to human PD-1. FIG. 4B showed binding to mouse PD-1. FIG. 4Cshowed binding to cynomolgus PD-1.

2. Cross-Reactivity to Human PD-1 Family Members CD28, CTLA4

Constructed cell lines that respectively express human PD-1, CD28,CTLA-4 or ICOS were transferred in to 96-well U-bottom plates (BD) at adensity of 2×10⁵ cells/well. Testing antibodies were diluted in washbuffer (1×PBS/1% BSA) and incubated with cells at 4° C. for 1 h. Afterwashing, the secondary antibody goat anti-human IgG Fc FITC (JacksonImmunoResearch Lab) was added and incubated at 4° C. in the dark for 1h. The cells were then washed once and resuspended in 1×PBS/1% BSA, andanalyzed by flow cytometery (BD) and FlowJo software.

FIG. 5 showed the result of cross-family test. The anti-PD-1 antibodiescan bind specifically to human PD-1, but not to CD28 and CTLA-4.

3. Blocking of Ligand Binding to PD-1,

3.1 The ability of anti-PD-1 antibodies to block PD-L1 binding to PD-1was tested by FACS as described in Example 2.3.3.2 The ability of anti-PD-1 antibodies to block PD-L2 binding to PD-1was tested by ELISA. Briefly, plates (Nunc) were coated with human PD-1at 1 μg/ml overnight at 4° C. Antibodies were serially diluted inblocking buffer and mixed with his tag conjugated PD-L2. After blockingand washing the coated plates, the antibody/PD-L2 mixture were added tothe plates, then incubated at room temperature for 1 h. The plates werethen washed and subsequently incubated with secondary antibody goatanti-his HRP (GenScript) for 1 h. After washing, TMB substrate was addedand the reaction was stopped by 2 M HCl. The absorbance at 450 nm wasread using a microplate reader (Molecular Device).

FIG. 6A showed the result of anti-PD-1 antibodies blocking human PD-L1binding to PD-1 transfected CHO-S cells. FIG. 6B shows the result ofanti-PD-1 antibodies blocking mouse PD-L1 binding to PD-1 transfected293F cells. FIG. 7 showed that the anti-PD-1 antibodies could blockhuman PD-L2 binding to PD-1 in a dose-dependent manner.

4. Full Kinetic Binding Affinity Tested by Surface Plasmon Resonance(SPR)

Antibodies were characterized for affinity and binding kinetics to PD-1by SPR assay using ProteOn XPR36 (Bio-Rad). Protein A protein (Sigma)was immobilized to a GLM sensor chip (Bio-Rad) through amine coupling.Purified antibodies were flowed over the sensor chip and captured by theProtein A. The chip was rotated 900 and washed with running buffer(1×PBS/0.01% Tween20, Bio-Rad) until the baseline was stable. Sevenconcentrations of human PD-1 and running buffer were flowed through thesensor chip at a flow rate of 30 μL/min for an association phase of 180s, followed by 300s dissociation. After regeneration, sevenconcentration of mouse PD-1 and running buffer were flowed through thesensor chip at a flow rate of 30 μL/min for an association phase of 180s, followed by 300 s dissociation. The chip was regenerated with pH 1.5H₃PO₄ after each run. The association and dissociation curve was fit by1:1 Langmuir binding model using ProteOn software.

Table.6A-6B showed the results of full kinetic binding affinity to humanand mouse PD-1 by SPR. WBP305BMK1 was synthesized according to the cloneof 5C4 from BMS patent U.S. Pat. No. 9,084,776B2. Keytruda was theanti-PD-1 drug from Merck. The same below. The results showed that theaffinity ability to human PD-1 by SPR assay was from 1.43E-8 to 5.64E-9mol/L. Comparing WBP305BMK1 with Keytruda, the K_(D) value of antibody2E5, 2G4 or 2C2 was much smaller, illustrating that 2E5, 2G4 or 2C2 hadbetter binding ability to human PD-1. In addition, the affinity abilityto mouse PD-1 was from 9.37E-9 to 3.89E-9 mol/L.

TABLE 6A Analyte Ligand ka (1/Ms) kd (1/s) KD (M) Chi² (RU²) U-value AhPD-1.His 1H6 6.44E+05 9.18E−03 1.43E−08 0.05 1 2E5 5.97E+05 3.66E−036.13E−09 0.14 1 2G4 6.63E+05 4.70E−03 7.09E−09 0.10 1 2C2 7.33E+054.14E−03 5.64E−09 0.03 1 W3052-16.88.z9- 3.82E+06 1.36E−01 3.56E−08 0.035 IgG4 (42720) WBP305BMK1 4.02E+05 1.35E−03 3.37E−09 0.01 1 Keytruda8.79E+05 2.28E−03 2.59E−09 0.07 1 B mPD-1.His 1H6 3.20E+05 3.00E−039.37E−09 0.06 1 2E5 3.23E+05 1.29E−03 3.99E−09 0.01 1 2G4 3.34E+051.30E−03 3.89E−09 0.01 1 2C2 2.21E+05 1.53E−03 6.92E−09 0.19 1W3052-16.88.z9- 1.95E+05 8.09E−03 4.16E−08 0.01 1 IgG4 (42720)

5. Binding Affinity of Anti-PD-1 Antibodies to Cell Surface PD-1Molecules Tested by Flow Cytometry (FACS)

CHO-S cells expressing human PD-1 or 293F cells expressing mouse PD-1were transferred in to 96-well U-bottom plates (BD) at a density of1×10⁵ cells/well. Testing antibodies were 1:2 serially diluted in washbuffer (1×PBS/1% BSA) and incubated with cells at 4° C. for 1 h. Thesecondary antibody goat anti-human IgG Fc FITC (3.0 moles FITC per moleIgG, (Jackson Immunoresearch Lab) was added and incubated at 4° C. inthe dark for 1 h. The cells were then washed once and resuspended in1XPBS/1% BSA, and analyzed by flow cytometery (BD). Fluorescenceintensity was converted to bound molecules/cell based on thequantitative beads (Quantum™ MESF Kits, Bangs Laboratories, Inc.). K_(D)was calculated using Graphpad Prism5.

Table.7A-7B show the results of binding affinity of anti-PD-1 antibodiesto cell surface human and mouse PD-1 molecules tested by flow cytometry.The results showed that the affinity ability to human PD-1 by FACS assaywas from 3.80E-10 to 2.15E-10 mol/L. In addition, the affinity abilityto mouse PD-1 was from 5.39E-8 to 1.74E-8 mol/L.

TABLE 7 Sample Best fit-KD (M) A 1H6 2.15E−10 2E5 2.30E−10 2G4 3.80E−102C2 2.64E−10 W3052-16.88.z9-IgG4 (42720) 4.32E−10 WBP305BMK1 2.62E−10Keytruda 1.79E−10 B 1H6 5.39E−08 2E5 2.90E−08 2G4 3.51E−08 2C2 1.74E−08W3052-16.88.z9-IgG4 (42720) 1.67E−08

6. Epitope Binning Test

The binding epitope of anti-PD-1 antibodies was compared with benchmarkantibody A and B by FACS. CHO-S cells expressing human PD-1 on the cellsurface were incubated with mixture of biotinylated benchmark antibody Aor B (1 μg/ml) and testing antibodies (serially diluted in wash buffer)at 4° C. for 1 h. The cells were washed and the second antibodyStreptavidin-PE were added and incubated for 30 min at 4° C. The cellswere then washed once and resuspended in 1×PBS/1% BSA, and analyzed byflow cytometery (BD).

FIG. 8A-8B showed the results of epitope binning assay suggesting thatthe anti-PD-1 antibodies are in the same or close epitope bin asbenchmark antibodies. FIG. 8A showed binning against WBP305BMK1 (U.S.Pat. No. 9,084,776). FIG. 8B showed binning against Keytruda (U.S. Pat.No. 8,168,757).

Furthermore, alanine scanning experiments on hPD-1 were conducted andtheir effect to antibody binding was evaluated. Alanine residues onhPD-1 were mutated to glycine codons, and all other residues weremutated to alanine codons. For each residue of the hPD-1 extracellulardomain (ECD), point amino acid substitutions were made using twosequential PCR steps. A pcDNA3.3-hPD-1_ECD.His plasmid that encodes ECDof human PD-1 and a C-terminal His-tag was used as template, and a setof mutagenic primer was used for first step PCR using the QuikChangelightning multisite-directed mutagenesis kit (Agilent technologies, PaloAlto, Calif.). Dpn I endonuclease was used to digest the parentaltemplate after mutant strand synthesis reaction. In the second-step PCR,linear DNA expression cassette which composed of a CMV promoter, anextracellular domain (ECD) of PD-1, a His-tag and a herpes simplex virusthymidine kinase (TK) polyadenylation was amplified and transientlyexpressed in HEK293F cells (Life Technologies, Gaithersburg, Md.).

Monoclonal antibodies W3052_r16.88.9 and Keytruda were coated in platesfor ELISA binding assay. After interacting with the supernatant thatcontains quantified PD-1 mutant or human/mouse PD-1_ECD.His protein(Sino Biological, China), HRP conjugated anti-His antibody was added asdetection antibody. Absorbance was normalized according to the averageof control mutants. After setting an additional cutoff to the bindingfold change (<0.55), the final determined epitope residues wereidentified.

The binding activities of the antibodies W3052_r16.88.9 and Keytruda toboth human and murine PD-1 were conducted (FIG. 9 ). W3052_r16.88.9 wasfound binding to both hPD-1 and mPD-1 while Keytruda only bound to thehuman one (FIG. 9 ). This unique functional cross-reactivity ofW3052_r16.88.9 can help provide more animal model options in preclinicalstudies when evaluating the drug safety. To explore the origin of theobserved binding behaviors, epitope mapping of both antibodies wereconducted.

Top 30 point-substituted hPD-1 mutants that significantly reducedantibody binding were shown in Table 8. Checking the positions of allthese residues on the hPD-1 crystal structures (PDB code 3RRQ and 4ZQK)revealed that some amino acids (e.g. Val144, Leul42, Val110, Met108,Cys123 etc.) were fully buried in the protein, and were unlikely todirectly contact any antibodies. The observed binding reductions mostprobably resulted from the instability or even collapse of hPD-1structure after alanine substitutions. According to the antigenstructure analysis, some of the residues don't involve binding activity,but are expected to respond to the stability of the hPD-1 structure,e.g. V144 and L142. Mutants that affect both antibodies were treated asfalse hot spots and were removed from the list. After setting anadditional cutoff to the binding fold change (<0.55), the finaldetermined epitope residues were listed in Table 9. They are 9 positionsto W3052_r16.88.9 and 5 positions to Keytruda.

Comparing the epitope residues of W3052_r16.88.9 and Keytruda in Table 9only revealed two overlapped hot spot residues. The rest looked quitediverse, which indicated that two antibodies might have adopted verydifferent mechanisms in terms of hPD-1 binding and hPD-L1 blocking.Reading the residue IDs in Table 9 is not straightforward to interpretthe mechanisms. All data in Table 9, as well as the hPD-L1 binding site,were therefore mapped on the crystal structure of hPD-1 to make a bettervisualization and comparison. (FIG. 10 ).

TABLE 8 The effect of PD-1 point mutations on antibody bindingW3052_r16.88.9 Keytruda PD-1 fold PD-1 fold #Residue change ^(a) SD#Residue change ^(a) SD V 144 0.09 0.01 P 89 0.18 0.02 L 142 0.21 0.01 D85 0.38 0.01 K 131 0.27 0.02 V 144 0.4 0.01 P 35 0.31 0 R 94 0.46 0.04 A129 0.34 0 F 106 0.47 0.05 V 64 0.34 0 K 78 0.48 0 P 83 0.38 0.03 P 830.5 0.01 L 128 0.39 0.01 D 92 0.5 0.02 S 137 0.42 0.01 P 39 0.54 0 F 950.42 0.01 A 81 0.57 0.01 P 130 0.44 0.01 C 123 0.57 0.01 C 123 0.44 0.01N 66 0.57 0.03 R 94 0.49 0.04 L 142 0.59 0.01 M 108 0.49 0.02 F 82 0.610.03 D 117 0.51 0.01 F 95 0.61 0.04 F 82 0.53 0.02 F 52 0.63 0.01 A 1320.54 0.02 M 108 0.64 0.06 V 110 0.54 0.02 L 128 0.68 0.01 N 49 0.55 0.01I 126 0.72 0.01 W 67 0.55 0.01 A 113 0.72 0.01 E 61 0.56 0.04 V 110 0.730.04 N 102 0.57 0.04 G 47 0.73 0.01 P 39 0.57 0.01 D 117 0.73 0.07 I 1260.59 0.04 N 49 0.73 0 A 113 0.6 0.01 S 87 0.74 0.06 F 52 0.61 0.02 L 420.76 0.01 H 155 0.62 0.04 N 102 0.76 0.01 R 86 0.64 0.08 W 67 0.81 0.01A 149 0.64 0 P 101 0.81 0.04 G 47 0.64 0.03 A 80 0.82 0.01 ^(a) Foldchange in binding is relative to the binding of several silent alaninesubstitutions.

TABLE 9 Identification of potential epitopes PD-1 to residue PD-1 toresidue r16.88.9 location Keytruda location P 35 A K 78 C′ V 64 C P 83C′ F 82 C′ D 85 C″ P 83 C′ P 89 C″ L 128 FG D 92 C″D A 129 FG P 130 FG K131 FG A 132 FG Cutoff: fold change <0.55 * The C″ strand observed onmPD-1 does not exist on hPD-1 structure. This β-sheet is replaced by astructreless loop on hPD-1. We still use C″ to label this region, justfor the purpose of easier comparison to mPD-1.

Two investigated antibodies W3052_r16.88.9 and Keytruda, although bothare functional in binding hPD-1 and blocking hPD-L 1, have obviouslydifferent epitopes (FIG. 10B, 10C). The epitope of Keytruda were mainlycontributed by the residues on the C′D loop (corresponding to the C″strand on mPD-1), which didn't intersect the PD-L1 binding site at all.This suggested the hPD-L1 blocking function of Keytruda relied more onits steric hindrance effects provided by the size of the antibody. Incontrast, the epitope mapping results show that the epitope of antibodyW3052_r16.88.9 was composed of hot spots distributed across multiplelocations, and have direct overlap with the hPD-L1 binding site (FIG.10A, 10B). W3052_r16.88.9 blocked hPD-L1 by means of competing withhPD-L1 in reacting to their common binding site. What's more,W3052_r16.88.9 had no interactions with the flexible C′D loop (or thecorresponding C″ strand on mPD-1), where human and murine PD-1 show bigstructural deviations (FIG. 11 ). Its binding site is mostly located onthe FG loop (Lin et al. (2008) PNAS 105: 3011-3016). That explains whyW3052_r16.88.9 can bind to both PD-1 species while Keytruda only bindsto the human one (FIG. 9 ). Because of this unique functionalcross-reactivity, the preclinical safety evaluations of W3052_r16.88.9could be conducted in mouse model, which will greatly simplify andaccelerate the development. Overall, antibody W3052_r16.88.9 is expectedto be more functional and developable than Keytruda.

7. In Vitro Function of Anti-PD-1 Antibodies Tested by Cell-Based Assays7.1 Mixed Lymphocyte Reaction (MLR) was Used to Test the Effects ofAnti-PD-1 Antibodies on T Lymphocytes Function

Human DCs, CD4⁺ T, CD8⁺ T and total T cells isolation: Human P3MCs werefreshly isolated from healthy donors using Ficoll-Paque PLUS (GE)gradient centrifugation. Monocytes were isolated using Human MonocyteEnrichment Kit (StemCell) according to the manufacturer's instructions.Cells were cultured in medium containing rhGM-CSF and rhIL-4 for 5 to 7days to generate dendritic cells. 18 to 24 hours before MLR, 1 μg/mL LPSwas added to the culture to induce the maturation of the DCs. Human CD4⁺T cells were isolated using Human CD4⁺ T Cell Enrichment Kit (StemCell)according to the manufacturer's protocol. Mouse CD4⁺ T cells wereobtained from the spleen of Balb/c mouse using Mouse CD4⁺ T CellIsolation Kit (StemCell) according to the manufacturer's protocol. MouseDCs were induced from bone marrow cells of C57BL/6 mouse in mediumcontaining rmGM-CSF and rmIL-4 for 5 to 7 days. 18 to 24 hours beforeMLR, 1 μg/mL LPS was added to the culture to induce the maturation ofthe DCs.

Briefly, primary dendritic cell (DC)-stimulated MLR was conducted in96-well, U-bottom tissue culture plates in 200 μL of RPMI 1640containing 10% FCS and 1% antibiotics. DCs were mixed with 1×10⁵ CD4⁺ Tcells at a ratio between 1:10 and 1:200 DC: T cells in the presence orabsence of testing antibodies or benchmark antibodies (form 166.75 nMdown to 0.00667 nM, generally total six concentrations). To determinethe effect of anti-PD-1 antibodies on T cell function, the cytokineproduction and T cell proliferation were determined. Results shown arerepresentative of a minimum of five experiments performed.

Cytokine detection: Human IFN-γ and IL-2 were measured by enzyme-linkedimmunosorbent assay (ELISA) using matched antibody pairs. The plateswere pre-coated with capture antibody specific for human IFN-γ (cat #Pierce-M700A) or IL-2 (cat # R&D-MAB602), respectively. Thebiotin-conjugated anti-IFN-γ antibody (cat # Pierce-M701B) or anti-IL-2antibody (cat # R&D-BAF202) was used as detecting antibody.

FIG. 12A showed anti-PD-1 antibodies increased IL-2 secretion in adose-dependent manner. FIG. 12B shows anti-PD-1 antibodies increaseIFN-γ secretion in a dose-dependent manner.

Proliferation assay: 311-thymidine (cat # PerkinElmer-NET027001MC) wasdiluted 1:20 in 0.9% NaCl solution, and added to the cell culture platesat 0.5 uCi/well. The plates were cultured in 5% CO₂ at 37° C. for 16 to18 hours, before the incorporation of 3H-thymidine into theproliferating cells was determined. FIG. 12C shows anti-PD-1 antibodiesincrease CD4⁺ T cells proliferation in a dose-dependent manner.

To determine the effect of anti-PD-1 antibodies on mouse T cellfunction, the cytokine production and mouse T cell proliferation weredetermined similarly. FIG. 13A-13C showed the results of mouse allo-MLRdemonstrating that the anti-PD-1 antibodies can enhance the function ofmouse CD4+ T cell. FIG. 13A showed anti-PD-1 antibodies increased IL-2secretion in a dose-dependent manner. FIG. 13B showed anti-PD-1antibodies increased IFN-γ secretion in a dose-dependent manner. FIG.13C showed anti-PD-1 antibodies increased CD4⁺ T cells proliferation ina dose-dependent manner.

7.2 Effect of Human Anti-PD-1 Antibodies on Cell Proliferation andCytokine Production by Autologous Antigen Specific Immune Response

In this assay, the CD4⁺ T cells and DCs were from a same donor. Briefly,CD4⁺ T cells were purified from PBMC and cultured in the presence of CMVpp⁶⁵ peptide and low dose of IL-2 (20 U/mL), at the meanwhile, DCs weregenerated by culturing monocytes from the same donor's PBMC in GM-CSFand IL-4. After 5 days, the CMV pp65 peptide treated CD4⁺ T cells wereco-cultured with DCs pulsed with CMV pp65 peptide in the absence orpresence of human anti-PD-1 antibodies or benchmark antibodies (ascontrol). On day 5, 100 μL of supernatants were taken from each ofcultures for IFN-γ measurement by ELISA as described above. Theproliferation of CMV pp65-specific T cells was assessed by 3H-thymidineincorporation as described above.

FIG. 14A-14B showed the results of human auto-MLR demonstrating theanti-PD-1 antibodies can enhance the function of human CD4⁺ T cell. FIG.14A showed anti-PD-1 antibodies increase IFN-γ secretion in adose-dependent manner. FIG. 14B showed anti-PD-1 antibodies increaseCD4⁺ T cells proliferation in a dose-dependent manner.

7.3 Effect of Human Anti-PD-1 Antibodies on Regulatory T Cell (Tregs)Suppressive Function

Tregs, a subpopulation of T cells, are a key immune modulator and playcritical roles in maintaining self-tolerance. Increased numbers of CD4⁺CD25⁺ Tregs were found in patients with multiple cancers and associatedwith a poorer prognosis. To determine whether the anti-PD-1 antibodiesaffect the immune suppressive role of Tregs, we compared the T cellfunction in the presence of Tregs with or without anti-PD-1 antibodytreatment. CD4⁺ CD25⁺ and CD4⁺ CD25-T cells were separated usingspecific anti-CD25 microbeads (StemCell) per manufacture's instruction.Two thousand mature DCs, 1×10⁵ CD4⁺CD25⁻ T cells, 1×10⁵ Treg cells andPD-1 antibodies were incubated in 96-well plates. The plates were keptat 37° C. in a 5% CO₂ incubator for 5 days. IFN-γ production andCD4⁺CD25⁻ cells proliferation were tested as described above.

FIG. 15 demonstrates that the anti-PD-1 antibodies can reverse thesuppressive function of Tregs. FIG. 15A showed anti-PD-1 antibodies canrestore the IFN-γ secretion. FIG. 15B showed anti-PD-1 antibodies canrestore the T-cell proliferation.

8. ADCC and CDC Test

PD-1 is expressed on variety of cell types. In order to minimizepotential toxicity to healthy PD-1 positive cells, the anti-PD-1antibodies were evaluated for their ability to mediateantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC).

8.1 ADCC Test

Human activated CD4⁺ T cells and various concentrations of PD-1antibodies were pre-incubated in 96-well plate for 30 minutes, and thenPBMCs were added at the effector/target ratio of 50:1. The plate waskept at 37° C. in a 5% CO₂ incubator for 6 hours. Target cell lysis wasdetermined by LDH-based cytotoxicity detection kit (cat #Roche-11644793001). The absorbance at 492 nm was read using a microplatereader (Molecular Device). Herceptin-induced SK-Br-3 cell lysis was usedas positive control.

FIG. 16 showed the result of ADCC test demonstrating the anti-PD-1antibodies did not mediate ADCC activity on activated CD4⁺ T cells.

8.2 CDC Test

Human activated CD4+ T cells and various concentrations of PD-1antibodies were mixed in 96-well plate. Human complement (Quidel-A112)was added at the dilution ratio of 1:50. The plate was kept at 37° C. ina 5% CO₂ incubator for 2 hours. Target cell lysis was determined byCellTiter-Glo. Rituxan®-induced Raji cell lysis was used as positivecontrol. The luminescence was read using a microplate reader (MolecularDevice).

FIG. 17 showed the result of CDC test demonstrating the anti-PD-1antibodies did not mediate CDC activity on activated CD4⁺ T cells.

Example 5: Treatment of In Vivo Tumor Model Using Human MonoclonalAntibodies Against PD-1 1. Experimental Design

TABLE 10 Grouping and dosing regimen of the in vivo animal efficacyexperiments of antibody 2E5 Dose- Volume Route of Frequency Treat- DoseParameters adminis- of adminis- group N¹ ment (mg/kg) (μl/g)² trationtration 1 6 Vehicle — 10 IP Q3D × 5 3 6 2E5 1 mg/kg 10 IP Q3D × 5 4 62E5 3 mg/kg 10 IP Q3D × 5 5 6 2E5 10 mg/kg  10 IP Q3D × 5 Annotations:¹N: mice number in each group ²Dose-Volume: 10 μL/g according to theweight of mouse. If the weight loss exceeds 15%, the dosing regimenshould be adjusted accordingly.

2. Methods 2.1 Cell Culture

Murine melanoma cell CloudmanS91 cell (ATCC-CCL-53.1) was cultured invitro as monolayer, and the culture condition was F-12K medium plus 2.5%FBS and 15% horse serum, 100 U/mL penicillin, and 100 μg/mLstreptomycin, incubate at 37° C. and 5% CO₂. The cells were digestedusing trypsin-EDTA and passaged twice a week routinely. Cells wereharvested, counted, and then inoculated when approximately 80%-90%confluent and the number is as required.

2.2 Injection of Tumor Cells 0.1 ml (5×10⁵ cells) Cloudmans91 cells wereinoculated subcutaneously in the right backside of each animal. When themean of tumor volume had reached approximately 64 mm³, theadministration started in groups. Grouping and dosing regimens wereshown in Table 10.

2.3 Tumor Testing and Index

Experimental index is to investigate whether the tumor growth wasinhibited, delayed or cured. Tumor diameters were measured with acaliper three times a week. Tumor volume is calculated using V=0.5a×b²,wherein a and b represents long and short diameters of the tumor,respectively.

Antitumor efficacy of the antibody was assessed by tumor growthinhibition TGI (%) or relative tumor proliferation rate T/C (%). TGI (%)reflected the rate of tumor growth inhibition. TGI (%) was calculated asfollows: TGI (%)=[(1−(average tumor volume at the end of administrationin the treatment group−average tumor volume at the start ofadministration in the treatment group))/(average tumor volume at the endof treatment in the solvent control group−average tumor volume at thestart of treatment in the solvent control group)]×100%.

Relative tumor proliferation rate T/C (%) was calculated as follows: T/C%=T_(RTV)/C_(RTV)×100% (T_(RTV): treatment group RTV; C_(RTV): negativecontrol group RTV). The relative tumor volume (RTV) was calculatedaccording to the results of tumor measurements using RTV=V_(t)/V₀,wherein V₀ was average tumor volume at the time of grouping (i.e., do),V_(t) was average tumor volume of a certain measurement; the data ofT_(RTV) and C_(RTV) were taken on the same day.

T-C (days) reflected tumor growth delay index, T represented averagedays passed when the tumor had reached a predetermined volume in thetreatment group (eg. 300 mm³), C represented the average days whentumors in the control group had reached the same volume.

Survival curves were plotted; animal survival time was defined as thetime from the administration to animal deaths or the time when tumorvolume had reached 2000 mm³. The median survival time (days) wascalculated in each group. Increased life span (ILS) was calculated bycomparison of the median survival times between the treated group andmodel control group and represented as a percentage over the lifetime ofthe model control group.

2.4 Statistical Analysis

The data including the average tumor volume at each time point in eachgroup and standard error (SEM) were analyzed statistically (refer toTable 11 for specific data). The experiment was completed on day 37after the administration; on day 13 after the administration, startsacrificing animals successively; and therefore the statistical analysisand evaluation for inter-group differences were based on the tumorvolume on day 13 after initiation of administration. For comparisonsbetween the two groups, data were analyzed using T-test; for comparisonsamong three or more groups, data were analyzed using one-way ANOVA. Ifstatistically significant difference was found for F value, data wereanalyzed using Games-Howell test. If no statistically significantdifference was found for F value, Dunnet (2-sided) test was then usedfor analysis. SPSS 17.0 was used for all data analysis. p<0.05 wasconsidered as significant difference. Survival time was analyzed usingKaplan-Meier method with the Log-rank test.

3. Results 3.1 Mortality, Morbidity and Body Weight Changes

Animal's weight is as an indirect reference for measurement of drugtoxicity. The impact of 2E5 on the weight of CloudmanS91 subcutaneoussyngeneic xenograft female DBA/2 mice model was as shown in FIG. 18 andFIG. 19 . In this model, all administration groups showed no significantweight loss (FIG. 18 ). Thus, 2E5 had no obvious toxicity in a mousemodel of melanoma CloudmanS91.

3.2 Tumor volume

Tumor volume in CloudmanS91 subcutaneously syngeneic xenograft femaleDBA/2 mouse model after 2E5 treatment was as shown in Table 11.

TABLE 11 Tumor volume at different times in each group Tumor volume(mm³)^(a) 2E5 2E5 2E5 Days Vehicle 1 mg/kg 3 mg/kg 10 mg/kg  0^(b) 66 ±9 65 ± 8 64 ± 8 63 ± 8 2 142 ± 23 129 ± 10 110 ± 10 94 ± 8 4 251 ± 39231 ± 34  162 ± 9B 143 ± 17 6 345 ± 65 339 ± 61 200 ± 13 197 ± 38 9 599± 66  597 ± 100 281 ± 38 291 ± 83 11  1,026 ± 173   943 ± 307 335 ± 66B475 ± 190  13  1,626 ± 262  1,089 ± 365  361 ± 81 B614 ± 273 Annotations: ^(a)average ± SEM; ^(b)Days after administration.

3.3 Tumor Growth Curve

Tumor growth curve was shown in FIG. 20 .

3.4 Antitumor Efficacy Evaluation

TABLE 12 Antitumor efficacy evaluation of 2E5 in CloudmanS91 Syngeneictumor model (based on the tumor volume on day 13 after initiation ofadministration) Tumor volume T-C (mm³)^(a) T/C ^(b) XTGI ^(b) (Days) pgroup (Day 13) (%) (%) (300 mm³) value ^(c) Vehicle 1,626 ± 262 — — — —2E5 (1 mg/kg) 1,089 ± 365 68.1 34.4 0 0.367 2E5 (3 mg/kg)  361 ± 81 22.981.0 5 0.008 2E5 (10 mg/kg)  614 ± 273 39.4 64.7 5 0.036 Annotations:^(a)average ± SEM; ^(b) Tumor growth inhibition was calculated by T/Cand TGI (TGI (%) = [1 − (T₁₃ − T₀)/(V₁₃ − V₀)] × 100); ^(c) p value wascalculated by tumor volume.

3.5 Survival Curves

Survival curves in each group were shown in FIG. 21 .

3.6 Survival Time

TABLE 13 Effect of 2E5 on survival of CloudmanS91 Syngeneic tumor modelMedian survival Prolonged survival Log Rank group time (Days) (%) Pvalue ^(a) Vehicle 16 — — 2E5 1 mg/kg 20  25 0.077 2E5 3 mg/kg N/A^(b)N/A 0.001 2E5 10 mg/kg 32 100 0.022 Annotations: ^(a) p-valuerepresented the comparison between the treatment group and the vehiclecontrol group; ^(b)At the end of the experiment, in 3 mg/kg group (2E5),the survival rate was 66.7%.

4. Discussion

In this study, we have evaluated the in vivo efficacy of 2E5 inCloudmanS91 syngeneic tumor model. Tumor volume in each group atdifferent time points were shown in Table 11, Table 12 and FIG. 20 ,survival time were shown in FIG. 21 and Table 13. On day 13 afteradministration, tumor volume of tumor-bearing mice in the solventcontrol group reached 1,626 mm³. A weak inhibitory effect was observedin 1 mg/kg 2E5 group compared with the control group, and the tumorvolume was 1,089 mm³ (T/C=68.1%, TGI=34.4%, p=0.367), tumor growth delaywas 0 days. A significant anti-tumor effect was observed in 3 mg/kg 2E5group compared with the solvent control group, and the tumor volume was361 mm³ (T/C=22.9%, TGI=81.0%, p=0.008), tumor growth delay was 5 days.A significant anti-tumor effect was also observed in 10 mg/kg 2E5 groupcompared with the solvent control group, the tumor volume was 614 mm³(T/C=39.4%, TGI=64.7%, p=0.036), tumor growth delay was 5 days.

In the experiment, the median survival time of tumor-bearing mice insolvent control group was 16 days. Compared with the vehicle controlgroup, the median survival time of tumor-bearing mice in 1 mg/kg 2E5group was 20 days, survival was prolonged 25% (p=0.077); survival rateof tumor-bearing mice in 3 mg/kg 2E5 group was 66.7% (p=0.001). Themedian survival time of tumor-bearing mice in 10 mg/kg 2E5 group was 32days, survival was prolonged 100% (p=0.022).

The changes in body weight of nude mice were shown in FIG. 19 . Goodtolerability of drug 2E5 has been found in all tumor-bearing mice, andno significant weight loss was observed in all treatment groups. Insummary, in this experiment, significant anti-tumor effects were shownin both 3 mg/kg group and 10 mg/kg group for CloudmanS91 subcutaneoussynergistic tumor model, which is not dose-dependent. Anti-tumor effectin 3 mg/kg group is better than that in 10 mg/kg group.

The description of the present invention has been made above by theexamples. However, it is understood by the skilled in the art that thepresent invention is not limited to the examples. The invention may beembodied in other specific forms without departing form the spirit oressential characteristics thereof. Scope of the invention is thusindicated by the appended claims rather than by the foregoingdescription, and all changes that come within the meaning and range ofequivalency of the claims are intended to be embraced therein.

1. An antibody or antigen binding fragment thereof that binds to anepitope of PD-1 comprising amino acids at positions 128, 129, 130, 131and 132 and at least one of amino acids at positions 35, 64, 82, 83 ofSEQ ID NO:
 24. 2. (canceled)
 3. The antibody or the antigen bindingfragment thereof of claim 1, wherein the murine PD-1 is mouse or ratPD-1.
 4. The antibody or the antigen binding fragment thereof of claim1, wherein the antibody a) binds to human PD-1 with a K_(D) of 2.15E-10M or less; and b) binds to mouse PD-1 with a K_(D) of 1.67E-08 M orless.
 5. The antibody of any of claim 1, wherein the antibody exhibitsat least one of the following properties: a) binds to human PD-1 with aK_(D) of between 4.32E-10 M and 2.15E-10 M and to mouse PD-1 with aK_(D) of between 5.39E-08 M and 1.67E-08 M; b) does not substantiallybind to human CD28, CTLA-4; c) increases T-cell proliferation; d)increases interferon-gamma production; or e) increases interleukin-2secretion.
 6. An antibody or an antigen binding fragment thereof,comprising an amino acid sequence that is at least 70%, 80%, 90% or 95%homologous to a sequence selected from a group consisting of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8 and 9, wherein the antibody specifically binds toPD-1.
 7. (canceled)
 8. The antibody, or an antigen-binding fragmentthereof claim 6, comprising: a) a variable region of a heavy chainhaving an amino acid sequence that is at least 70%, 80%, 90% or 95%homologous to a sequence selected from a group consisting of SEQ ID NOs:1 and 2; and b) a variable region of a light chain having an amino acidsequence that is at least 70%, 80%, 90% or 95% homologous to a sequenceselected from a group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9,wherein the antibody specifically binds to PD-1.
 9. The antibody or anantigen binding fragment thereof of claim 8, comprising: a) a variableregion of a heavy chain having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1 and 2; and b) a variable region of alight chain having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and 9, wherein the antibodyspecifically binds to PD-1.
 10. The antibody or an antigen bindingfragment thereof of claim 6, comprising a complementarity-determiningregion (CDR) having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 10-23, wherein the antibody specifically bindsto PD-1.
 11. The antibody, or antigen-binding fragment thereof gf claim10, comprising: a heavy chain variable region comprising CDR1, CDR2, andCDR3 sequences; and a light chain variable region comprising CDR1, CDR2,and CDR3 sequences, wherein the heavy chain variable region CDR3sequence comprises a sequence selected from a group consisting of SEQ IDNOs: 12 and 13, and conservative modifications thereof, wherein theantibody specifically binds to PD-1.
 12. The antibody of claim 10,wherein the light chain variable region CDR3 sequence comprises an aminoacid sequence selected from a group consisting of SEQ ID NOs: 20, 21, 22and 23, and conservative modifications thereof.
 13. The antibody ofclaim 10, wherein the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from a group consisting ofamino acid sequences of SEQ ID NO: 11, and conservative modificationsthereof.
 14. The antibody of claim 10, wherein the light chain variableregion CDR2 sequence comprises an amino acid sequence selected from agroup consisting of amino acid sequences of SEQ ID NO: 19, andconservative modifications thereof.
 15. The antibody of claim 10,wherein the heavy chain variable region CDR1 sequence comprises an aminoacid sequence selected from a group consisting of amino acid sequencesof SEQ ID NO: 10, and conservative modifications thereof.
 16. Theantibody of claim 10, wherein the light chain variable region CDR1sequence comprises an amino acid sequence selected from a groupconsisting of amino acid sequences of SEQ ID NOs: 14, 15, 16, 17 and 18,and conservative modifications thereof.
 17. The antibody of claim 6,wherein the antibody is a chimeric or humanized or human antibody. 18.The antibody of claim 6, wherein the antibody exhibits at least one ofthe following properties: a) binds to human PD-1 with a K_(D) of2.15E-10 M or less and to mouse PD-1 with a K_(D) of 1.67E-08 M or less;b) does not substantially bind to human CD28, CTLA-4; c) increasesT-cell proliferation; d) increases interferon-gamma production; or e)increases interleukin-2 secretion.
 19. A nucleic acid molecule encodingthe antibody, or the antigen binding fragment of claim
 6. 20. A cloningor expression vector comprising the nucleic acid molecule of claim 19.21. A host cell comprising one or more cloning or expression vectors ofclaim
 20. 22. A process for the production of the antibody of claim 6,comprising culturing the host cell of claim 21 and isolating theantibody.
 23. The process of claim 22, wherein the antibody is preparedthrough immunization in SD rat with human PD-1 extracellular domain andmouse PD-1 extracellular domain.
 24. A transgenic mouse comprising humanimmunoglobulin heavy and light chain transgenes, wherein the mouseexpresses the antibody of claim
 6. 25. A hybridoma prepared from themouse of claim 24, wherein the hybridoma produces said antibody.
 26. Apharmaceutical composition comprising the antibody, or the antigenbinding fragment of claim 6, and one or more of a pharmaceuticallyacceptable excipient, diluent or carrier.
 27. An immunoconjugatecomprising the antibody, or antigen-binding fragment thereof, accordingto claim 6, linked to a therapeutic agent.
 28. A pharmaceuticalcomposition comprising the immunoconjugate of claim 27 and apharmaceutically acceptable excipient, diluent or carrier.
 29. A methodfor preparing an anti-PD-1 antibody or an antigen-binding fragmentthereof comprising: a) providing: (i) a heavy chain variable regionantibody sequence comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1 and 2; ; and/or (ii) a light chainvariable region antibody sequence comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8 and9, ; and b) expressing the antibody sequence as a protein.
 30. A methodof modulating an immune response in a subject comprising administeringto the subject the antibody, or antigen binding fragment of claim
 6. 31.(canceled)
 32. A method of inhibiting growth of tumor cells in asubject, comprising administering to the subject a therapeuticallyeffective amount of the antibody, or the antigen-binding fragment ofclaim 6 to inhibit growth of the tumor cells.
 33. The method of claim32, wherein the tumor cells are of a cancer selected from a groupconsisting of melanoma, renal cancer, prostate cancer, breast cancer,colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer,cancer of the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, and rectal cancer.
 34. The method ofclaim 32, wherein the antibody is a chimeric antibody or humanizedantibody.